US 2015O125571 A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0125571 A1 PURKAYASTHA et al. (43) Pub. Date: May 7, 2015

(54) FERMENTED DAIRY PRODUCTS Related U.S. Application Data CONTAINING SWEETENER AND FLAVOR MODIFIER DERVED FROM STEVA AND (60) Provisional application No. 61/881,030, filed on Sep. METHODS OF PRODUCING SAME 23, 2013. (71) Applicant: PURECIRCLE USA INC., Oak Brook, Publication Classification IL (US) (51) Int. Cl. (72) Inventors: Siddhartha PURKAYASTHA, A23C 9/13 (2006.01) Lombard, IL (US): Avetik A23C 9/23 (2006.01) MARKOSYAN, Yerevan (AM); Marcia (52) U.S. Cl. PETIT, Chicago, IL (US); Kristina CPC ...... A23C 9/1307 (2013.01); A23C 9/123 CHKHAN, Kuala Lumpur (MY): (2013.01) Mariam ADAMYAN, Bandar Enstek (MY) (57) ABSTRACT Sweetener compositions including mainly highly purified (21) Appl. No.: 14/494,322 Steviol glycosides, and methods for making and using these compositions as a Sweetener to Sweeten fermented dairy (22) Filed: Sep. 23, 2014 products, are described. Patent Application Publication May 7, 2015 Sheet 1 of 4 US 2015/O125571 A1

Effect of sweeteners on sensory profile

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Patent Application Publication May 7, 2015 Sheet 2 of 4 US 2015/O125571 A1

Fig.2 Whitemass Evaluation Sugar Control

Sweet intensity

Creaminess

Sourness 8% Sugar control before : incubation : Bitterness : :

Bitter 8.8% Sugar control after incubation Aftertaste :

Sweet Aftertaste

Overaiking

FG. 3 Whitemass Evaluation Alpha/SG-95

Sweet intensity

Creaminess

Sourness 200/300 ppm Alpha/SG 95 before incubation Bitterness

Bitter Aftertaste 200/300 ppm Alpha/SG 95 after incubation : Sweet Aftertaste

Overal Liking Patent Application Publication May 7, 2015 Sheet 3 of 4 US 2015/O125571 A1

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Patent Application Publication May 7, 2015 Sheet 4 of 4 US 2015/O125571 A1

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FERMENTED DARY PRODUCTS of carbohydrate residues at the C13 and C19 positions. The CONTAINING SWEETENER AND FLAVOR leaves of Stevia are able to accumulate up to 10-20% (on dry MODIFIER DERVED FROM STEVA AND weight basis) Steviol glycosides. The major glycosides found METHODS OF PRODUCING SAME in Stevia leaves are Rebaudioside A (2-10%), Stevioside (2-10%), and Rebaudioside C (1-2%). Other glycosides such BACKGROUND OF THE INVENTION as Rebaudioside B, D, E, and F. M(RebX), Steviolbioside and 0001 1. Field of the Invention rubusoside are found at much lower levels (approx. 0-0.2%). 0002 The present invention relates generally to Sweetener 0006 Steviol glycosides differ from each other not only by compositions comprising mainly highly purified Steviol gly molecular structure, but also by their Sweetness intensity and cosides and methods for making and using them as a Sweet taste quality or properties as shown in the Table 1 (Reference: ener to Sweeten fermented dairy products. In particular, the PCT/US2012/030210). TABLE 1.

Steviol Glycoside Molecules and their sweetening

Sweetening power Steviol glycosides R1 (C-19) R2 (C-13) relative to sucrose Taste Quality

Stevioside (G3) B-Glc B-Glc-B-Glc(2-1) 150-300 O Rebaudioside A (G4) B-Glc B-Glc-B-Glc(2-1)- 200-400 +2 B-Glc(3-1) Rebaudioside B (G3) H B-Glc-B-Glc(2-1)- 300-350 O B-Glc(3-1) Rebaudioside C (G4) B-Glc B-Glc-C-Rha (2-1)- SO-120 -1 B-Glc(3-1) Rebaudioside D (G5) B-Glc-B-Glc(2-1) B-Glc-B-Glc(2-1)- 200-300 +3 B-Glc(3-1) Rebaudioside E (G4) B-Glc-B-Glc(2-1) B-Glc-B-Glc(2-1) 2SO-300 +1 Rebaudioside F (G4) B-Glc B-Glc-B-Xyl(2-1)- Na Na B-Glc(3-1) Rubusoside(G2) B-Glc B-Glc 110 -2 Steviolmonoside(G1) H B-Glc Na Na Steviolbioside(G2) H B-Glc-B-Glc(2-1) 100-12S -3 DulcosideA (G3) B-Glc B-Glc-C-Rha (2-1) SO-120 -2

*G# means number of carbohydrate residue (mainly glucose) present invention relates to different Sweetener compositions 0007. However, even in a highly purified state, Steviol derived from Stevia extracts comprising non-caloric or low glycosides still possess undesirable taste attributes such as caloric natural high-potency Sweetener and glucosylated Ste bitterness, sweet aftertaste, licorice flavor, etc. One of the viol glycosides as a flavor modifier. The present invention main obstacles for the Successful commercialization of Stevia also relates to Sweetener compositions and method to incor sweeteners are these undesirable taste attributes. It was shown porate the Sweetener in dairy application that can improve the that these flavor notes become more prominent as the con taste of the reduced Sugar dairy products by imparting a more centration of steviol glycosides increases (Prakash I. DuBois Sugar-like taste or characteristic. In particular, the Sweetener G. E., Clos J. F., Wilkens K. L., Fosdick L. E. (2008) Devel compositions and methods provide a more Sugar-like tempo opment of Rebiana, a natural, non-caloric Sweetener. Food ral profile in dairy applications. Chem. Toxicol., 46, S75-S82.). 0003 2. Description of the Related Art 0008. The glucosylated Steviol glycosides (GSG) can be made by attaching additional glucose units to Steviol glyco 0004. Nowadays sugar alternatives are receiving increas side molecules by selected enzyme(s). The GSG molecules ing attention due to awareness of many diseases in conjunc show improved Sweetness profile with significant improve tion with consumption of high-calorie foods and beverages. ment of non-sweet taste (U.S. Pat. Nos. 4,219,571, 7,838,044, Consumers are also looking for natural ingredients in their and 7,807.206). Depending on the degree of glucosylation food and beverages, thus non-caloric Sweeteners of natural (number of glucose units added), the Sweetness intensity can origin are becoming increasingly popular. The Sweet herb be significantly reduced and GSG can act as a flavor modifier Stevia Rebaudiana Bertoni produces a number of diterpene and enhances sweetness profile (Glucosylated Steviol Glyco glycosides which feature high intensity Sweetness and sen sides as flavor modifiers, US Patent application, PCT/ sory properties Superior to those of many other high potency US2011/033737, FEMA GRAS 26, FEMAH4728). SWeetenerS. 0009. If the taste profile of high-potency sweeteners 0005. The above-mentioned sweet glycosides, have a (HPS) could be modified to impart desired taste characteris common aglycon, Steviol, and differ by the number and type tics, it can contribute to taste characteristics in low calorie US 2015/O125571 A1 May 7, 2015

food products that are more desirable for consumers. This process is critical to achieve the desired taste and Sweetness invention combines different natural Sweeteners, especially profile. This innovation, in part, also pertains to the process to Stevia sweeteners in certain proportion to provide better and prepare fermented products sweetened with Stevia rounded sweetness profile with limited non-sweet taste pro Sweetener, where addition of Sweetener during the process is file. This invention also show that when blend of such sweet critical to achieve the consistent taste and Sweetness profile eners is combined with very small amount of GSG, which over the shelf life. acts as a flavor modifier, an unexpectedly rich and rounded Sweetness profile is achieved in food applications, particu SUMMARY OF THE INVENTION larly in fermented dairy products like yogurt. 0010 Yogurt is made from milk and bacterial culture 0014. The present invention is aimed to overcome the dis added with sugars, stabilizers, fruits and flavors. The com advantages of existing Stevia Sweeteners in fermented dairy mercial manufacturing process includes standardizing formulation. The present invention is directed to a taste and (modifying) the composition of and pasteurizing the milk; flavor profile improving composition. The invention fermenting at warm temperatures; cooling to develop struc describes blends of selected Steviol glycosides at a certain ture (white mass); stirring the white mass and adding fruit, ratios for producing a high purity food ingredient from the Sugar and other ingredients. Raw milk composition is stan extract of the Stevia Rebaudiana Bertoni plant and use thereof dardized to get total solid content around 16% with 1-5% fat in various fermented or cultured dairy products as a Sweetness and 11-14% solids-not-fat (SNF). The compositional goal is and flavor modifier. Non-limiting examples of fermented or achieved by removing milk fat and either by evaporating cultured dairy products include cheese, yogurt, yogurt drink, excess water or by adding milk Solid or concentrated milk. acidophilus milk, sour creme, , , , , After standardization, stabilizers are added and milk is pas vili, filmjolk, creme cheese, amasi, , calpis, , teurized, which involves heating the milk to a relatively high , clabber, dadhia, and other similar cultured dairy prod temperature and holding it there for a set amount of time. The uctS. milk is then homogenized to break the fat globules to smaller 0015 The invention, in part, pertains to an ingredient com size and then cooled down to 43-46° C. before adding fer prising Steviol glycosides of Stevia Rebaudiana Bertoni mentation culture. The inoculated milk is held at warm tem plant. The Steviol glycosides are selected from the group perature for several hours to reach desired lactic acid level consisting of stevioside, Rebaudioside A, Rebaudioside B, with minimum acidity of 0.9% and pH about 4.4. The yogurt Rebaudioside C, Rebaudioside D, Rebaudioside E. Rebau is cooled to achieve the firmer texture. For flavored yogurt, dioside F, dulcoside A, Steviolbioside, rubusoside, as well as flavors, fruit prep or Sweeteners are added to prepared yogurt other steviol glycosides found in Stevia Rebaudiana Bertoni mixing thoroughly, then packaged and stored in refrigerated plant and mixtures thereof. In addition, the invention pertains temperature. to an ingredient comprising Steviol glycosides and glucosy 0011 with different flavors and fruit puree have lated Steviol glycosides to impart Superior taste and flavor become very popular, where such additives have more than profile in cultured dairy products. 50% sugar in them. With the trend toward healthy eating, 0016. The invention also pertains to an ingredient com many manufacturers offer a low Sugar and low fat version of prising Stevia derived Sweeteners and phosphate salt of yogurts. Low or no Sugar yogurts are often Sweetened with Sodium or calcium ion, especially mono-Sodium phosphate aspartame. With ever-increasing market-demand for natural that is known to act as a buffering agent in different food and ingredients in food, Stevia Sweeteners are deemed to be per beverage. However, the mono-Sodium phosphate salt is Sur fect solution, if stevia could deliver the sugar-like taste that prisingly discovered to modify the Sweetness profile in the consumers expect in fermented dairy products like yogurt. fermented sweetened with stevia sweetener. 0012 Prakash et al (Food Chem Toxicol, 56, 2008) 0017. The invention also directed to a process of incorpo reported that stevia sweetener (Rebiana or Reb A) was stable rating Sweetener composition during the making of yogurt. during the processing and storage of yogurt. The publication The sweetener and flavor are generally added to the white states that “no significant loss (of Stevia Sweetener) was mea mass after the fermentation of the milk. Those skilled in the sured during (190°F. for 5 min) and fermen art would assume that the change of the order of addition of tation (for making plain yogurt). Rebiana was stable through Sweetener during the manufacturing process would have no out a 6-week storage period (40°F). The main challenge effect on the final product. However we surprisingly discov with the Stevia Sweeteners like Reb Ain yogurt application is ered that unlike to common belief, the introduction of Stevia that it modifies the flavor and acidity profile and leave non Sweetener prior to fermentation yield a product completely sweet taste of stevia in fermented dairy products. Addition different in taste profile. Additionally, we discovered that the ally, the sweetness profile of yogurt product with Stevia introduction of stevia sweetener prior to fermentation yield a Sweetener may change and provide impression of less Sweet product with consistent sweetness profile over the typical ness after certain period of shelf life. shelf life of the yogurt. 0013 This innovation, in part, pertains to the formulation of the sweetened with specific (0018. Within the description of this invention we will blends of natural Sweeteners and flavors derived from Stevia show that, when certain blend of steviol glycosides are added extract. The innovation, in part, also pertains to the formula to Sweeten the yogurt before fermentation rather after fer tion of the fermented mil products containing a buffering mentation, the Sweetness profile improves significantly and agent that Surprisingly provides the Smooth and rounded remains consistent over the shelf life. sweetness profile infermented milk products sweetened with 0019. This invention will also show the method of com Stevia Sweeteners. This innovation, in part, pertains to the bining stevia-derived sweeteners and flavor modifier such as process to prepare fermented milk products Sweetened with GSG in no-added Sugar yogurt applications contribute Supe Stevia sweetener, where addition of sweetener during the rior taste profile. US 2015/O125571 A1 May 7, 2015

BRIEF DESCRIPTION OF THE DRAWINGS trol sample contained Sugar and monosodium phosphate. Table 3 shows the sweetener amount in the samples and lists 0020 FIG. 1 is a graph showing the effect of sweeteners on the comments from a trained sensory panel of 5 members. The yogurt sensory profile. monosodium phosphate provided buffering of acid profile 0021 FIG. 2 is a graph showing the results of an evaluation and contributed some Smoothness of texture, as expected. of a white mass made with Sugar. However, the enhancement of sweetness by the interaction of 0022 FIG.3 is a graph showing the results of an evaluation phosphate salt with Stevia Sweetener was an unexpected out of a white mass made with Alpha/SG-95. come in the formulation. 0023 FIG. 4 is a graph showing the results of an evaluation the sweet aftertaste of a white mass made with SG-95 and Alpha. TABLE 3 0024 FIG.5 is a graph showing the results of an evaluation Effect of mono-sodium phosphate with stevia Sweetener of the sweet aftertaste of a white mass made with SG-95 and On Sensory attributes of White mass. NSF-02. Sugar SG95 NSFO2 Phosphate 0025 FIG. 6 is a graph showing the results of an evaluation (%) (ppm) (ppm) (ppm) Sensory Comments of the sweetness intensity of a white mass made with Alpha Control 8 458 More Sour, more and SG-95. lactic acid 0026 FIG. 7 is a graph showing the results of an evaluation Sample 1 627 125 458 Sweeter than control, of the sweetness intensity of a white mass made with SG-95 Smoother, slight and NSF-02. bitter, off note Sample 2 627 125 Less Sweet, Harsh, high lactic, high dairy EXAMPLE1 note, Sour milk taste Preparation of White Mass (Yogurt) 0027 Milk was brought to boil and stevia ingredient or EXAMPLE 3 Sugar (Table 2) was dissolved in a measured quantity of milk as it was cooling down. The milk was cooled downto 37C and Formulation with Different Combination of monosodium phosphate was added (46 mg per 100 ml of Sweeteners Added Before Incubation milk). Adequate amount (5-10 g/100 ml) of starter (yogurt 0029. The white mass samples containing different sweet culture) was added with moderate stirring and then the mix ener system (Table 4) were prepared by the methodology ture was kept in the incubator at 37 C for 16 hours. The white outline in Example 1. The refrigerated samples were tested by mass is then kept at a refrigerated temperature (4C) for 24 a trained sensory panel to investigate the effect of Sweeteners hours and served cold for tasting. on the taste of the white mass.

TABLE 2 TABLE 4 Sweetener and flavor modifier used for making the white mass Effect of Sweeteners on the Sensory profile of white mass Sweeteners. Mfg. Description of the Sweeteners Sugar Alpha + SG95 - Flavors Company and flavor modifier Control Reb A Alpha SG95 NSFO2 Sugar Domino Cane Sugar granulated Test No. Reb A97 PureCircle Highly purified Rebaudioside A, purity >97%, Ingredients (g) C 1 2 3 4 Stevia extract SG95 PureCircle Blend of steviol glycosides extracted from Sugar 28 stevia leaf Purity >95%; Reb A content >50% Alpha O.152 O.33 PC Alpha PureCircle Proprietary blend of steviol glycosides; Reb SG95 O.22 0.175 A content >75% Reb A O.152 NSFO2 PureCircle Glucosylated Steviol glycosides (GSG), a NSFO2 O.0437 flavor modifier Whole milk 332.5 360.35 360.35 1132.45 360.28 *All stevia sweeteners meet JECFA (2010) spec; NSF02 is a natural flavor complex derived from stewia extract Total (g) 36O.S 36O.S 36O.S 1133 36O.S Initial white mass samples were made with two commercial The sensory panel of 8 members found the sweetness profile starters (Stonyfield Farm and Dannon) and a small group of of the white mass is much more rounded for the SG95/Alpha panel members tasted both samples. Panel preferred the white (Test 3) blend than RebA and Alpha sweetened samples. The mass made with Stonyfield Farm culture, which gave white panel found significant improvement of bitter and bitter after mass with Smooth texture, less acid and slightly Sweeter taste. taste with the blend of SG95/Alpha Sweeteners compared to RebA and Alpha. The panel also found the overall liking and EXAMPLE 2 sweet aftertaste improved in Test 3 as show in FIG. 1. Effect of Phosphate Salt on Sensory Attributes EXAMPLE 4 0028. To determine the effect of mono-sodium phosphate on the taste and texture of white mass, test samples were Effect of the Addition of Sweeteners. During the prepared using the methodology outlined in example 1 with Preparation of Yogurt and without monosodium phosphate and Sweetened with 0030. To determine the effect of addition of sweeteners SG95 and NSF02 (a flavor modifier) combination. The con before and after incubation, white mass samples were made US 2015/O125571 A1 May 7, 2015

using the methodology outlined in Example 1. The Sweeten Results: ers tested were sugar and a blend of Alpha and SG95 as outlined in Table 5. A sensory panel of 8 members evaluated 0034. The addition of stevia or stevia and flavor before those samples for Sweet intensity, Sourness, creaminess, bit incubation had a lower Sweet-aftertaste compare to terness, bitter aftertaste, Sweet aftertaste and overall liking as sample when Stevia or stevia and flavor was added after shown in FIG. 2 and FIG. 3. incubation. 0035. The sweet-aftertaste consistently decreased over the shelf life of 2 weeks. TABLE 5 Effect of the addition of Sweeteners during EXAMPLE 6 the preparation of the White mass Effect of the Addition of Sweeteners and Flavors Sugar Alpha + Control SG95 During the Preparation of Yogurt on Sweetness Over Test Number Shelf-Life Ingredients (g) C 3 0036. To determine the effect of the addition of sweeteners and flavors before and after incubation on Sweetness during Sugar 28 Alpha O.1OS shelf-life, white mass samples were made using the method SG95 O.O7 ology outlined in Example 1. The sweeteners tested were Reb A sugar and a blend of Alpha, SG95 and NSF-02 as outlined in NSFO2 Table 6. A sensory panel of 4 members evaluated those Whole milk 332.5 360.33 samples for Sweet intensity, and Sweet aftertaste as shown in Total (g) 36O.S 36O.S FIG. 6 and FIG. 7. Results: Results: 0037. The control samples (with sugar) showed no change in the sweetness perception over the shelf life of 0031 Stevia added before incubation had lower sweet 14 days aftertaste compare to sample when Stevia was added 0038. The addition of stevia sweetener combination or after incubation. the combination stevia sweetener and flavor (GSG) 0032 Control sample of sugar added before and after before incubation provided consistent sweetness profile incubation did not show any significant difference in over 14 days. The variation is within the experimental attributes variation. 0039. The addition of stevia sweetener combination or EXAMPLE 5 the combination of Stevia sweetener and flavor (GSG) Effect of the Addition of Sweeteners and Flavors after incubation provided steadily decreasing Sweetness During the Preparation of Yogurt perception after 6-8 days of shelf life. 0040. The addition of the selected Stevia sweetener 0033. To determine the effect of addition of sweeteners blend or the blend of sweetener and flavor (GSG) before and flavors before and after incubation, white mass samples incubation had a lower Sweet aftertaste during a 14 day were made using the methodology outlined in Example 1. period of time compared to sample when those blends The Sweeteners tested were sugar and a blend of Alpha, SG95 were added after incubation. and NSF-02 as outlined in Table 6. A sensory panel of 4 0041. The addition of stevia sweetener blend or the members evaluated those samples for Sweet aftertaste as blend of sweetener and flavor (GSG) before incubation shown in FIG. 4, and FIG. 5. had a higher Sweet intensity after 14 days compared to samples when when those blends were added after incu TABLE 6 bation.

Effect of the addition of Sweeteners and flavor 1. A blend of natural sweetener composition derived from during the preparation of the White mass Stevia extract and a phosphate salt provide Superior taste and sweetness profile in fermented dairy product. Formulation 2. The natural sweetener composition of claim 1, wherein NAS Stevia NAS Stevia said purified Stevia extract comprises a blend of Steviol gly Target sweetened Sweetened cosides with Reb Acontent more than 50% andless than 95%, Original SG95/Alpha SG95.NSF-02 more specifically between 60 and 90% of total steviol glyco Milk 3.5% Fat 85 88 88 sides present in the Sweetener. Natural Yoghurt 5 5 5 3. The natural sweetener composition of claim 2, wherein Sucrose 5 O O said other steviol glycosides are less than 50 percent of a SMP 3 5 5 Double Cream 2 2 2 blend of primarily Reb B. Reb D, Reb C and other steviol SG95 O.08 O.O23 glycosides. Alpha O16 4. The natural sweetener composition of claim 1, wherein NSF-02 O.O12S the phosphate salts, especially mono-Sodium phosphate is Total 1OO 100 1OO used as the sweetness profile modifier. 5. The natural sweetener composition of claim 1, wherein glucosylated Steviol glycoside is used as a flavor modifier, US 2015/O125571 A1 May 7, 2015

where the concentration of GSG is between 10 to 500 ppm, more specifically between 50 and 300 ppm in the final prod uct. 6. The natural Sweetener composition that consists of the sweetener of claim 1 and natural flavor of claim 5 to provide better Sweetness and aftertaste in dairy products. 7. The process for the preparation of a natural Sweetener composition, which comprising the steps of formulating a natural Sweetener composition; dry mixing the natural Sweet ener composition; granulated or agglomerated to a certain particle size distribution; 8. A process of making yogurt, where in the Sweetener composition of claim 1 is added before incubation rather after fermentation and setting of the yogurt. 9. The dairy product made with Sweetener composition of claim 2 with process condition of claim 2 results in better and consistent Sweetness and reduced Sweetness aftertaste. 10. The dairy product made with Sweetener composition of claim 2 with process condition of claim 9 results in better and consistent Sweetness and reduced Sweetness aftertaste in dairy product.